My team and I have ordered bacteriophages specific to L. monocytogenes from ATCC and have been attempting to find the titer of them for months now with little to no success. We are unsure if it is just that the bacteriophages are not especially effective, or if we are doing somethign wrong (or both). We're having the following main problems:

-How to grow the phage to maximize them? We've tried concentrating a 48hr liquid culture of bacteria and then incubating with phage for another 48 hours. This still doesn't seem to have worked.

-Unsure what % agar to make the semisolid. 0.5-0.8% seems too runny, 1-1.2% could prevent the bacteria and phage from mixing. Each batch seems to come out differently as well. 

-How to spread the bacteria? Some procedures say to use a spreader, others say to add it to the semisolid before pouring that on the plate.

-Can dilutions of phage be stored overnight at 4°C and used again? Or will the phage decay?

-Is it possible that ATCC sent us dud phage?

Please if you can answer even one of these questions it would help a lot! Thank you

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